title: Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha): biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns.
abstract: Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ~60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[(14)C]-sucrose to orphan shoot (twigs) and [(14)C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.
authors.affiliation: CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh, India.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Kushwaha
authors.forename: Neelam Singh
authors.initials: NS
authors.lastname: Sangwan
authors.forename: Prabodh Kumar
authors.initials: PK
authors.lastname: Trivedi
authors.forename: Arvind Singh
authors.initials: AS
authors.lastname: Negi
authors.forename: Laxminarain
authors.initials: L
authors.lastname: Misra
authors.forename: Rajender Singh
authors.initials: RS
authors.lastname: Sangwan
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D053898:Biosynthetic Pathways / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D015971:Gene Expression Regulation, Enzymologic; D017343:Genes, Plant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics*; D010802:Phylogeny; D010936:Plant Extracts; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014326:Tropanes / Q000378:metabolism*; D032341:Withania / Q000201:enzymology* / Q000235:genetics*
chemical_list: D010936:Plant Extracts; D011994:Recombinant Proteins; D014326:Tropanes; C005864:tropine; D000429:Alcohol Oxidoreductases; C091007:tropine dehydrogenase; C030693:Ashwagandha